The human temporomandibular joint is susceptible to osteoarthritis, rheumatoid arthritis, fractures, ankylosis, and dysfunctional syndromes, collectively affecting over 10 million individuals in the U.S. alone. The current clinical gold standards for surgical replacement of the mandibular condyle such as bone/cartilage grafts and artificial materials suffer from substantial deficiencies such as donor site defects, limited supply, immunorejection, and transmission of pathogens. In response to NIDCR's RFA, the overall objective of the present proposal is to regenerate the mandibular condyle of the temporomandibular joint using mesenchymal stem cells seeded in biocompatible polymers. Specific Aim 1, will optimize the densities of adult bone marrow-derived mesenchymal stem cells (MSCs). Once extracted from mouse tibiofemoral bones, MSCs will be expanded in cell culture, induced to differentiate into osteoprogenitor and chondroprogenitor cells and seeded in hydrogels shaped in prefabricated molds of human mandibular condyles. The regenerative outcome of tissue-engineered mandibular condyles including architecture, chondrogenesis and osteogenesis will be evaluated qualitatively and quantitatively by our published methods such as selective histologic labels, immunohistochemistry of bone and cartilage markers, computerized histomorphometry, and micromechanical testing with atomic force microscopy. In Specific Aim 2, multilayered osteochondral constructs will be fabricated in the shape of human mandibular condyle, and implanted in the dorsum of immunodeficient mice. Substrartified osteochondral constructs are anticipated to mimic normal condylar development. Specific Aim 3 will focus on functional enablement of tissue-engineered osteochondral constructs. In vitro hydraulic micromechanical stresses will be applied to osteochondral constructs shaped in human mandibular condyle ex vivo so that they experience simulated functional environments. In Specific Aim 4, autologous adult bone-marrow derived mesenchymal stem cells will be extracted from the rabbit tibia by needle aspiration, seeded in hydrogel scaffolds in the shape of rabbit mandibular condyle. After up to 16 weeks of surgical implantation of rabbit mandibular condyles in autologous rabbits, the harvested, tissue-engineered mandibular condyles will be implanted to replace surgically created, unilateral mandibular condyle defects in each autologous rabbit. Normal masticatory functions will be allowed for up to 16 weeks followed by biochemical, histomorphometric and biomechanical analyses of the tissue-engineered mandibular joint. The anticipated findings may have implications in the ultimate fabrication of functional, tissue-engineered human mandibular condyles using autologous human stem cells for clinical implantation.